Rare occurrence of severe blindness and deafness in Friedreich ataxia: a case report

A Potential New Therapeutic Approach for Friedreich Ataxia: Induction of Frataxin Expression With TALE Proteins

Friedreich's Ataxia Induced Pluripotent Stem Cells Model Intergenerational GAA⋅TTC Triplet Repeat Instability

150 years of Friedreich Ataxia: from its discovery to therapy

Introduction: Friedreich ataxia (FRDA) is a recessive neurodegenerative disease characterized by progressive ataxia, dyscoordination, and loss of vision. The variable length of the pathogenic GAA triplet repeat expansion in the FXN gene in part explains the interindividual variability in the severity of disease. The GAA repeat expansion leads to epigenetic silencing of FXN; therefore, variability in properties of epigenetic effector proteins could also regulate the severity of FRDA. Methods: In an exploratory analysis, DNA from 88 individuals with FRDA was analyzed to determine if any of five non-synonymous SNPs in HDACs/SIRTs predicted FRDA disease severity. Results suggested the need for a full analysis at the rs352493 locus in SIRT6 (p.Asn46Ser). In a cohort of 569 subjects with FRDA, disease features were compared between subjects homozygous for the common thymine SIRT6 variant (TT) and those with the less common cytosine variant on one allele and thymine on the other (CT). The biochemical properties of both variants of SIRT6 were analyzed and compared. Results: Linear regression in the exploratory cohort suggested that an SNP (rs352493) in SIRT6 correlated with neurological severity in FRDA. The follow-up analysis in a larger cohort agreed with the initial result that the genotype of SIRT6 at the locus rs352493 predicted the severity of disease features of FRDA. Those in the CT SIRT6 group performed better on measures of neurological and visual function over time than those in the more common TT SIRT6 group. The Asn to Ser amino acid change resulting from the SNP in SIRT6 did not alter the expression or enzymatic activity of SIRT6 or frataxin, but iPSC-derived neurons from people with FRDA in the CT SIRT6 group showed whole transcriptome differences compared to those in the TT SIRT6 group. Conclusion: People with FRDA in the CT SIRT6 group have less severe neurological and visual dysfunction than those in the TT SIRT6 group. Biochemical analyses indicate that the benefit conferred by T to C SNP in SIRT6 does not come from altered expression or enzymatic activity of SIRT6 or frataxin but is associated with changes in the transcriptome.

The role of the mitochondrial protein frataxin in iron storage and detoxification, iron delivery to iron-sulfur cluster biosynthesis, heme biosynthesis, and aconitase repair has been extensively studied during the last decade. However, still no general consensus exists on the details of the mechanism of frataxin function and oligomerization. Here, using small-angle x-ray scattering and x-ray crystallography, we describe the solution structure of the oligomers formed during the iron-dependent assembly of yeast (Yfh1) and Escherichia coli (CyaY) frataxin. At an iron-to-protein ratio of 2, the initially monomeric Yfh1 is converted to a trimeric form in solution. The trimer in turn serves as the assembly unit for higher order oligomers induced at higher iron-to-protein ratios. The x-ray crystallographic structure obtained from iron-soaked crystals demonstrates that iron binds at the trimer-trimer interaction sites, presumably contributing to oligomer stabilization. For the ferroxidation-deficient D79A/D82A variant of Yfh1, iron-dependent oligomerization may still take place, although >50% of the protein is found in the monomeric state at the highest iron-to-protein ratio used. This demonstrates that the ferroxidation reaction controls frataxin assembly and presumably the iron chaperone function of frataxin and its interactions with target proteins. For E. coli CyaY, the assembly unit of higher order oligomers is a tetramer, which could be an effect of the much shorter N-terminal region of this protein. The results show that understanding of the mechanistic features of frataxin function requires detailed knowledge of the interplay between the ferroxidation reaction, iron-induced oligomerization, and the structure of oligomers formed during assembly.

You have accessThe ASHA LeaderFeature1 Dec 2009Audiologists' Role in Early Diagnosis of Usher Syndrome Josara WallberAuD, CCC-A Josara Wallber Google Scholar , AuD, CCC-A https://doi.org/10.1044/leader.FTR4.14162009.5 SectionsAbout ToolsAdd to favorites ShareFacebookTwitterLinked In Usher syndrome is a frequent cause of deaf-blindness and involves sensorineural hearing loss and retinitis pigmentosa (RP). It occurs in four of every 10,000 births (NIH). The hearing loss is always sensorineural; the vision loss is always progressive; and the etiology of this combination is always genetic, specifically autosomal recessive. Parents are literally "blindsided" by this diagnosis and frequently complain this cannot be genetic because it is "not in their family." Such is the nature of recessive inheritance: both parents are asymptomatic, unknowing carriers of the same gene. RP is a degenerative retinal disease in which initially sighted individuals gradually lose their vision. Although RP occurs in one of every 4,000 persons in the general population, about 14% of all RP cases are caused by Usher syndrome (Hamel, 2006). The pattern of vision loss is quite predictable: night blindness followed by restricted tunnel vision and later loss of acuity and color perception. The onset and rate of progression of RP are, however, highly variable. The delayed onset of expression presents a challenge to the audiologist in assisting families with the diagnosis of Usher syndrome. Although clinical observation and behavioral measures of visual function are possible in older children, the definitive diagnostic test is objective: an electroretinalgram (ERG). Using electrodes placed on the cornea, an ERG measures the responses of retinal photoreceptor cells to light stimulation. There are three recognized clinical types of Usher syndrome, labeled simply Type I, Type II, and Type III. While some atypical expressions have been identified, they are beyond the scope of this article (for a review, see Cohen et al., 2007). The three types are distinguished primarily by severity of hearing loss, presence or absence of vestibular function, and onset of vision loss. All these variables are important, as hearing loss alone cannot diagnose Usher syndrome, nor can it differentiate clearly between the subtypes (Wagenaar et al., 1996). Characteristics Type I presents with congenital, severe-to-profound sensorineural hearing loss, no vestibular function, and noticeable vision involvement in the preteen years. Due to the severity of the hearing loss, hearing aids are generally ineffective. Individuals with Type I are typically identified as deaf during childhood and, prior to cochlear implantation, placed in educational programs that focus on visual communication. Because of the lack of vestibular function these children demonstrate delays in sitting and walking, with the latter being reported at older than 18 months of age (Moller et al., 1989). Night blindness typically appears by age 10. These children may be afraid of the dark and are often described as clumsy because they bump into or trip over objects in the environment. Significant deterioration of visual field and acuity begins between the second and third decade of life, with cataracts being a common complication (Piazza et al., 1986; Edwards et al., 1998; Sadeghi, 2006). Most individuals with Type II exhibit a stable, moderate sensorineural hearing loss (Reisser et al., 2002) and often respond well to amplification. The sub-Type IIa, however, may demonstrate progressive hearing loss not found in other Type II expressions (Sadeghi et al., 2004). Balance is unaffected and vision loss is unnoticed until late teens. Although the sequence of RP progression is similar to that of other types of RP (Iannaccone et al., 2004), visual field and acuity impairments are somewhat less severe compared to Type I during the third and fourth decades of life (Piazza et al., 1986; Edwards et al., 1998; Sadeghi, 2006). Type III has the most variable onset and presentation. Hearing loss is progressive and vestibular function may or may not be affected (Sadeghi et al., 2005). This clinical type, although common in Finland, is rare in the United States and has vision outcomes more similar to Type I (Plantinga, 2006). Type III represents 40% of all cases in Finland but only 2%–4% of all cases in the United States. The National Center for Hearing Assessment and Management (NCHAM) reports that 95% of infants born in the United States are evaluated prior to discharge by early hearing detection and intervention programs (EHDI), which have lowered the average age of identification of hearing loss from 12–18 months to 6 months or younger (Harrison & Roush, 1996). Sininger and colleagues (2009) demonstrated that newborns screened through EHDI are diagnosed more than 24 months earlier than those babies who were not screened. Despite these achievements, a diagnosis of Usher syndrome, with its devastating vision prognosis, typically lags five or 10 years behind the identification of the hearing loss (Kimberling & Lindenmuth, 2007). Although parents learn of their child's hearing loss relatively early, without a differential diagnosis they make critical decisions related to intervention, communication, and educational options without knowing their child eventually will be blind. Parents rely upon audiologists for information and support in working with their children who have hearing loss. ASHA's Guidelines for the Audiologic Assessment of Children From Birth to 5 Years of Age recommend that audiologists should "as appropriate, discuss additional specialty evaluations (e.g., genetics, ophthalmology, child development) with parents/caregivers and the infants' primary care provider" (ASHA, 2004, p. 13). This recommendation requires that the audiologist be familiar with genetic epidemiology of hearing loss and resources for referral and information. Once a child is identified, intervention services require a multi-disciplinary team, but audiologists are often the first—and primary—health care provider for individuals with Usher syndrome, and they must recognize and refer for proper and timely diagnosis those individuals who present with clinical signs and symptoms that may suggest the presence of Usher syndrome. Access Audiology Focuses on Usher Syndrome For more information on Usher syndrome, visit the November/December issue of Access Audiology, the bi-monthly clinical e-newsletter that highlights topics of relevance to audiologists. This issue includes Usher syndrome-related resources on the ASHA Web site as well as other Web resources. Anyone may subscribe to Access Audiology by sending a blank e-mail with "subscribe" in the subject line to [email protected]. References American Speech-Language-Hearing Association (2004) Guidelines for the audiologic assessment of children from birth to 5 years of age. Available from www.asha.org/policy. Google Scholar Cohen M., Bitner-Glindziez M., & Luxon L. (2007). The changing face of Usher syndrome: Clinical implications.International Journal of Audiology, 46, 82–93. Google Scholar Edwards A., Fishman G., Anderson R., Grove S., & Derlackie D. (1998). Visual acuity and visual field impairment in usher syndrome.Archives of Ophthalmology, 116(2), 165–168. Google Scholar Hamel C. (2006). Retinitis pigmentosa.Orphanet Journal of Rare Diseases, 1(40), 1–12. Google Scholar Harrison S., & Rousch J. (1996). Age of suspicion, identificaton, and intervention for infants and young children with hearing loss: a national study.Ear and Hearing, 17, 55–62. Google Scholar Innaccone A., Kritchevsky S., Ciccarelli M., Tedesco S., Macahuso C., Kimberling W., & Somes G. (2004). Kinetics of visual field loss in usher syndrome type II.Investigative Ophthalmology & Visual Science, 45(3), 784–792. Google Scholar Kimberling W., & Lindenmuth A. (2007). Genetics, hereditary hearing loss, and ethics.Seminars in Hearing, 28(3), 216–225. Google Scholar Moller C., Kimberling W., Davenport S., Priluck L., & White V. (1989). Usher syndrome: An otoneurologic study.Laryngoscope, 99, 73–79. Google Scholar National Institute of Health. (2008, February). Usher Syndrome, National Institute on Deafness and Other Communication Disorders athttp://www.nidcd.nih.gov/health/hearing/usher.asp Accessed October 2009. Google Scholar National Center for Hearing Assessment and Management. (2009). Available athttp://www.infanthearing.org. Google Scholar Piazza L., Fishman G., Farer M., Derlacki D., & Anderson R. (1986). Visual acuity loss in patients with usher's syndrome.Archives of Ophthalmology, 104(9), 1336–1339. Google Scholar Plantinga R., Pennings R., Huygen P., Sankila E., Tuppurainen K., Kleemola L., Cremer C., & Deutman A. (2006). Visual impairment in Finnnish usher syndrome type III.Aca Ophthalmologica Scandinavica, 84(1), 36–41. Google Scholar Reisser C., Kimberling W., & Otterstedde C. (2002). Hearing loss in usher syndrome type II is nonprogressive. Annals of Otology, Rhinology, and Laryngology, 111, 1108–111. Google Scholar Sadeghi M, Cohn E., Kelly W., Kimberling W., Tranebjarg L., & Moller C. (2004). Audiological findings in usher syndrome types IIa and II (non-IIa).International Journal of Audiology, 43, 136–143. Google Scholar Sadeghi M., Cohn E., Kimberling W., Tranebjarg L., & Moller C. (2005). Audiological and vestibular features in affected subject with USH3: a genotype/phenotype correlation.International Journal of Audiology, 44, 307–316. Google Scholar Sadeghi A., Eriksson K., Kimberling W., Sjostrom A., & Moller C. (2006). Longterm visual prognosis in usher syndorme types 1 and 2.Aca Ophthalmologica Scandinavica, 84(4), 537–544. Google Scholar Sininger Y., Martinez A., Eisenberg L., Christensen E., Grimes A., & Hu J. (2009). Newborn hearing screening speeds diagnosis and access to intervention by 20–25 months.Journal of the American Academy of Audiology, 20, 49–57. CrossrefGoogle Scholar Wagenaar M., Snik A., Kimberling W., & Cremers C. (1996). Carriers of usher syndrome type IB: is audiometric identification possible?.The American Journal of Otology, 17, 853–858. Google Scholar Author Notes Josara Wallber, AuD, CCC-A, spent 25 years at the National Technical Institute for the Deaf, where in addition to her work with aural rehabilitation, amplification, and cochlear implants for college students, she taught in the deaf education program and was a certified ophthalmic assistant working closely with the deaf-blind community. She is an associate clinical professor at Idaho State University, where she teaches and supervises clinical activities with an emphasis on cochlear implants. Contact her at [email protected]. Advertising Disclaimer | Advertise With Us Advertising Disclaimer | Advertise With Us Additional Resources FiguresSourcesRelatedDetails Volume 14Issue 16December 2009 Get Permissions Add to your Mendeley library History Published in print: Dec 1, 2009 Metrics Current downloads: 567 Topicsasha-topicsleader_do_tagasha-article-typesleader-topicsCopyright & Permissions© 2009 American Speech-Language-Hearing AssociationLoading ...

Friedreich ataxia (FRDA) is caused by reduced frataxin (FXN) concentrations. A clinical diagnosis is typically confirmed by DNA-based assays for GAA-repeat expansions or mutations in the FXN (frataxin) gene; however, these assays are not applicable to therapeutic monitoring and population screening. To facilitate the diagnosis and monitoring of FRDA patients, we developed an immunoassay for measuring FXN. Antibody pairs were used to capture FXN and an internal control protein, ceruloplasmin (CP), in 15 μL of whole blood (WB) or one 3-mm punch of a dried blood spot (DBS). Samples were assayed on a Luminex LX200 analyzer and validated according to standard criteria. The mean recovery of FXN from WB and DBS samples was 99%. Intraassay and interassay imprecision (CV) values were 4.9%-13% and 9.8%-16%, respectively. The FXN limit of detection was 0.07 ng/mL, and the reportable range of concentrations was 2-200 ng/mL. Reference adult and pediatric FXN concentrations ranged from 15 to 82 ng/mL (median, 33 ng/mL) for DBS and WB. The FXN concentration range was 12-22 ng/mL (median, 15 ng/mL) for FRDA carriers and 1-26 ng/mL (median 5 ng/mL) for FRDA patients. Measurement of the FXN/CP ratio increased the ability to distinguish between patients, carriers, and the reference population. This assay is applicable to the diagnosis and therapeutic monitoring of FRDA. This assay can measure FXN and the control protein CP in both WB and DBS specimens with minimal sample requirements, creating the potential for high-throughput population screening of FRDA.

Inherited deficiency in the mitochondrial protein frataxin (FXN) causes the rare disease Friedreich's ataxia (FA), for which there is no successful treatment. We identified a redox deficiency in FA cells and used this to model the disease. We screened a 1600-compound library to identify existing drugs, which could be of therapeutic benefit. We identified the topical anesthetic dyclonine as protective. Dyclonine increased FXN transcript and FXN protein dose-dependently in FA cells and brains of animal models. Dyclonine also rescued FXN-dependent enzyme deficiencies in the iron–sulfur enzymes, aconitase and succinate dehydrogenase. Dyclonine induces the Nrf2 [nuclear factor (erythroid-derived 2)-like 2] transcription factor, which we show binds an upstream response element in the FXN locus. Additionally, dyclonine also inhibited the activity of histone methyltransferase G9a, known to methylate histone H3K9 to silence FA chromatin. Chronic dosing in a FA mouse model prevented a performance decline in balance beam studies. A human clinical proof-of-concept study was completed in eight FA patients dosed twice daily using a 1% dyclonine rinse for 1 week. Six of the eight patients showed an increase in buccal cell FXN levels, and fold induction was significantly correlated with disease severity. Dyclonine represents a novel therapeutic strategy that can potentially be repurposed for the treatment of FA.

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disease caused by insufficient expression of frataxin (FXN), a mitochondrial iron-binding protein required for Fe-S cluster assembly. The development of treatments to increase FXN levels in FRDA requires elucidation of the steps involved in the biogenesis of functional FXN. The FXN mRNA is translated to a precursor polypeptide that is transported to the mitochondrial matrix and processed to at least two forms, FXN(42-210) and FXN(81-210). Previous reports suggested that FXN(42-210) is a transient processing intermediate, whereas FXN(81-210) represents the mature protein. However, we find that both FXN(42-210) and FXN(81-210) are present in control cell lines and tissues at steady-state, and that FXN(42-210) is consistently more depleted than FXN(81-210) in samples from FRDA patients. Moreover, FXN(42-210) and FXN(81-210) have strikingly different biochemical properties. A shorter N terminus correlates with monomeric configuration, labile iron binding, and dynamic contacts with components of the Fe-S cluster biosynthetic machinery, i.e. the sulfur donor complex NFS1·ISD11 and the scaffold ISCU. Conversely, a longer N terminus correlates with the ability to oligomerize, store iron, and form stable contacts with NFS1·ISD11 and ISCU. Monomeric FXN(81-210) donates Fe(2+) for Fe-S cluster assembly on ISCU, whereas oligomeric FXN(42-210) donates either Fe(2+) or Fe(3+). These functionally distinct FXN isoforms seem capable to ensure incremental rates of Fe-S cluster synthesis from different mitochondrial iron pools. We suggest that the levels of both isoforms are relevant to FRDA pathophysiology and that the FXN(81-210)/FXN(42-210) molar ratio should provide a useful parameter to optimize FXN augmentation and replacement therapies.

Friedreich's ataxia (FRDA) is a rare hereditary neurodegenerative disorder caused by a GAA repeat expansion in the FXN gene. There is still no cure or quantitative biomarkers reliaby correlating with the progression rate and disease severity. Investigation of functional and structural alterations characterizing white (WM) and gray matter (GM) in FRDA are needed prerequisite to monitor progression and response to treatment. Here we report the results of a multimodal cross-sectional MRI study of FRDA including Voxel-Based Morphometry (VBM), diffusion-tensor imaging (DTI), functional MRI (fMRI), and a correlation analysis with clinical severity scores. Twenty-one early-onset FRDA patients and 18 age-matched healthy controls (HCs) were imaged at 3T. All patients underwent a complete cognitive and clinical assessment with ataxia scales. VBM analysis showed GM volume reduction in FRDA compared to HCs bilaterally in lobules V, VI, VIII (L>R), as well as in the crus of cerebellum, posterior lobe of the vermis, in the flocculi and in the left tonsil. Voxel-wise DTI analysis showed a diffuse fractional anisotropy reduction and mean, radial, axial (AD) diffusivity increase in both infratentorial and supratentorial WM. ROI-based analysis confirmed the results showing differences of the same DTI metrics in cortico-spinal-tracts, forceps major, corpus callosum, posterior thalamic radiations, cerebellar penduncles. Additionally, we observed increased AD in superior (SCP) and middle cerebellar peduncles. The WM findings correlated with age at onset (AAO), short-allelle GAA, and disease severity. The intragroup analysis of fMRI data from right-handed 14 FRDA and 15 HCs showed similar findings in both groups, including activation in M1, insula and superior cerebellar hemisphere (lobules V–VIII). Significant differences emerged only during the non-dominant hand movement, with HCs showing a stronger activation in the left superior cerebellar hemisphere compared to FRDA. Significant correlations were found between AAO and the fMRI activation in cerebellar anterior and posterior lobes, insula and temporal lobe. Our multimodal neuroimaging protocol suggests that MRI is a useful tool to document the extension of the neurological impairment in FRDA.

The number of GAA repeats in the FXN gene is a major but not sole determinant of the clinical presentation of Friedreich ataxia (FRDA). The objective of this study was to establish whether the length of the GAA repeat tract in the FGF14 gene, which is associated with another neurodegenerative disorder (SCA27B), affects the clinical presentation (age at onset, mFARS score) of patients with FRDA. The number of GAA repeats in the FXN and FGF14 genes was determined using PCR in a cohort of 221 patients with FRDA. Next, we compared absolute lengths of the FGF14 GAAs with FXN GAAs, followed by correlative analyses to determine potential effects of FGF14 GAA length on age at onset and clinical presentation (mFARS) of FRDA. We found no significant correlation between the size of the GAA repeats in FXN and FGF14 loci in our FRDA cohort. Moreover, the number of GAAs in FGF14 did not affect the clinical presentation of FRDA even in a small number of cases where a long FGF14 allele was present. Despite both molecular and clinical similarities between FRDA and SCA27B, the length of the GAA repeats in the FGF14 gene, including potentially pathogenic alleles, did not influence the clinical presentation of FRDA.

Friedreich ataxia (FA) is an autosomal recessive disease characterized by progressive damage to the nervous system and severe cardiac abnormalities. The disease is caused by a GAA•TTC triplet repeat expansion in the first intron of the FXN gene, resulting in epigenetic repression of FXN transcription and reduction in FXN (frataxin) protein which results in mitochondrial dysfunction. Factors and pathways that promote FXN repression represent potential therapeutic targets whose inhibition would restore FXN transcription and frataxin protein levels. Here, we performed a candidate-based RNAi screen to identify kinases, a highly druggable class of proteins, that when knocked down upregulate FXN expression. Using this approach, we identified Rho kinase ROCK1 as a critical factor required for FXN repression. ShRNA-mediated knockdown of ROCK1, or the related kinase ROCK2, increases FXN mRNA and frataxin protein levels in FA patient-derived induced pluripotent stem cells (iPSCs) and differentiated neurons and cardiomyocytes to levels observed in normal cells. We demonstrate that small molecule ROCK inhibitors, including the FDA-approved drug belumosudil and fasudil, reactivate FXN expression in cultured FA iPSCs, neurons, cardiomyocytes, and FA patient primary fibroblasts and ameliorate the characteristic mitochondrial defects in these cell types. Remarkably, treatment of transgenic FA mice of both sexes with belumosudil or fasudil upregulates FXN expression, ameliorates the mitochondrial defects in the brain and heart tissues, and improves motor coordination and muscle strength. Collectively, our study identifies ROCK kinases as critical repressors of FXN expression and provides preclinical evidence that FDA-approved ROCK inhibitors may be repurposed for treatment of FA.

The Yeast Connection to Friedreich Ataxia

Since the discovery of the DNA double helix, major advances in biology have been; the development of recombinant DNA technology in the 1970s, methods to amplify DNA and gene targeting technology in the late 1980s. In organisms such as yeast and mice, the ability to accurately add or delete genetic information transformed biology, allowing an unmatched level of precision in studies of gene function. But, the ability to easily and specifically edit the genetic material of other cells and organisms remained impossible until recently for molecular biologists. The recent advent of programmable nucleases has dramatically changed the efficiency and speed of genome manipulation in several model organisms including cultured cells, as well as whole animals and plants. These tools opened up a powerful technique for biology research now called “genome editing” or “genome engineering” (Carroll, 2011; Hsu et al., 2014; Kim and Kim, 2014). In the first half of my doctoral studies, I developed genome-editing strategies to discover drug targets for a rare genetic disease called Friedreich’s Ataxia. Friedreich’s Ataxia (FRDA) is a neurodegenerative disease caused by deficiency of the mitochondrial protein frataxin (FXN) (Campuzano et al., 1997). This deficiency results from an expansion of a trinucleotide GAA repeat in the first intron of the FXN gene (Campuzano et al., 1996; Durr et al., 1996). Therapeutics that reactivate FXN gene expression are expected to be beneficial to FRDA patients (Gottesfeld, 2007). However, high-throughput screening (HTS) for FXN activators has so far met with limited success because current cellular models do not accurately assess endogenous FXN gene regulation. Here I used genome-editing technologies to generate a cellular model in which a luciferase reporter is introduced into the endogenous FXN locus. Using this system in a high-throughput genomic screen, we discovered novel inhibitors of FXN-luciferase expression. I confirmed that reducing expression of one of these inhibitors, PRKD1, led to an increase in FXN expression in FRDA patient fibroblasts (Villasenor et al., 2015). We then used reprogramming technologies to create a disease-relevant situation and test small molecules that specifically modulate PRKD1. We found that WA-21-JO19, a chemical inhibitor of PRKD1, increases FXN expression levels in iPSC-derived FRDA patient neurons. This approach, developed at the interface between academic and pharmaceutical research, demonstrates how a combination of genome editing, cellular reprogramming, and high-throughput biology can generate an effective novel drug discovery platform. In the second part of my doctoral work, we developed an interface between genome editing and proteomics to isolate native protein complexes produced from their natural genomic contexts. In many biological processes, proteins act as members of protein complexes. Understanding the molecular composition of protein complexes is a key task towards explaining their function in the cell. Conventional affinity purification followed by mass spectrometry analysis is a broadly applicable method to decipher molecular interaction networks and infer protein function. However, traditional affinity purification methods are limited by a number of factors such as antibody specificity and are sensitive to perturbations induced by overexpressed target proteins. Here, we combined genome editing with tandem affinity purification to circumvent current limitations. I uncovered subunits and interactions among well-characterized complexes and report the isolation of novel Mettl3-binding partners. The multi-protein complex composed of two active methyltransferases Mettl3 and Mettl14 mediates methylation of adenosines at position N6 on RNA molecules (Bokar et al., 1994; Bokar et al., 1997; Liu et al., 2014). N6-methyladenosine is the most abundant internal modification in eukaryotic mRNA and is often found on introns, which implies that methylation occurs co-transcriptionally (Fu et al., 2014). My work identified a set of nuclear RNA binding proteins, which specifically interact with the Mettl3-Mettl14 complex. We are currently testing the ability of these factors to function as “recruiters” of the Mettl3-Mettl14 complex to nascent mRNAs in the cell nucleus. In summary, our approach solidly establishes how a combination of genome editing and proteomics can simplify explorations of protein complexes as well as the study of post-translational modifications. In addition, this approach opens up new opportunities to study native protein complexes in a wide variety of cells and model organisms and will likely enable the systematic investigation of mammalian proteome function.

Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients' CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich's ataxia.